Cellular Characteristics of an Asbestos (Chrysotile-B) Resistant Subline of an HTLV-1-Immortalized Human Polyclonal T Cell Line (MT-2)

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1 Table of Contents Cellular Characteristics of an Asbestos (Chrysotile-B) Resistant Subline of an HTLV-1-Immortalized Human Polyclonal T Cell Line (MT-2) PL-4-5 Takemi Otsuki Takemi Otsuki, Yoshie Miura, Akiko Takata-Tomokuni, Fuminori Hyodoh, Hironobu Katsuyama* Department of Hygiene, Department of Public Health*, Kawasaki Medical School, Japan Introduction There are two common subtypes of pneumoconiosis, silicosis and asbestosis. Patients with silicosis have been exposed to silica and their condition is often complicated by autoimmune disorders such as SLE or SSc (systemic scleroderma) 1-3. Asbestosis patients have been exposed to asbestos (silicates) in such forms as chrystotile and crocidolite, and may develop not only respiratory diseases but also malignant tumors, including lung cancers and malignant mesotheliomas 4-7. We have been focusing our investigations on the immunological abnormalities found in silicosis and have found abnormalities of various molecules in the Fas-mediated apoptotic pathway 8-14 and detected various unique autoantibodies These facts and our findings indicate that silica and silicates affect the human immune system and may cause dysregulation of autoimmunity and reduction of tumor immunity. To explore the immunological cellular and molecular effects of silicates, an HTLV-1 immortalized human polyclonal T cell line, MT-2 19, was employed. Appearance of Apoptosis Following Short and High Dose Exposure Before the long-term and low-dose exposure effects of chrysotile on MT-2 cells could be examined in an in vitro model of human asbestosis from the immunological viewpoint, it was necessary for us to clarify what occurs after short and relatively high-dose exposure to chrysotile in MT-2 cells. Therefore, MT-2 cells were cultured with or without 5 to 5 µg/ml of chrysotile-a (CA) for one to four days and cell growth, the appearance of apoptosis (TUNEL method), activation of caspases 3 and 9, the relative balance of Bcl2 and Bax proteins, the release of cytochrome-c to cytoplasma from mitochondria, activation of the p38 and JNK MAP kinases, which stimulate the apoptotic signal, and production of superoxide were analyzed. As shown in Fig.1, MT-2 cells cultured with CA exhibited growth inhibition (Fig. 1-A), and increases in apoptotic fractions (Fig. 1-B and C) in a dose dependent manner. The appearance of apoptosis was also confirmed by the detection of activated caspase 3 and caspase 9 (Fig 2), which are the typical indicators of activation of the mitochondrial apoptotic pathway. Thereafter, molecular modifications known to take place in the mitochondrial apoptotic pathway were examined by Western blotting. As shown in Fig. 3, cytochrome-c was released from mitochondria (Mi) to cytoplasma (Cy), the relative expression ratio of BAX/Bcl2 increased, and JNK and p38, both known as apoptosis-inducing MAK kinases, were phosphorylated in a dose-dependent manner in MT-2 cells cultured with, 1, and 25 mg/ml of CA.

2 A Cell number (x 1 3 /dish) Cell number B Culture days TUNEL-positive Conc. of Ch rysotile-a (µg/ml) 1 C Apoptotic Fraction (%) Fig. 1 [A] Growth curve of MT-2 cells cultured with various concentrations of chrysotile-a (CA). [B] TUNEL detection of apoptotic cells in MT-2 cells cultured with CA and increase of its fraction in a dose-dependent manner [C].

3 Cell number A B C Activated Caspase Activated Caspase 3 (%) Day 2 Day 3 Caspase 3 Caspase precursor cleaved precursor cleaved Fig. 2 [A] Activation of caspase 3 analyzed by flow cytometry. [B] Increase of activated caspase 3 fraction in dose and time-dependent manners when MT-2 cells were cultured with various concentrations of CA. [C] Activation of caspase 3 and 9 analyzed by Western blotting. Cleaved forms were observed in a dose-dependent manner.

4 1 2 A Cy Mi Cy Mi Cy Mi.5 Cytochrome C 1 25 Relative ratio of cytochrome C (Cy/Mi) B Relative ratio of BAX/Bcl2 Bcl-2 BAX 1 25 C 1 25 Relative ratio of Relative ratio of p-p38/p38 p-jnk/jnk JNK 2 pjnk 3.5 p p-p Fig. 3 Western blotting analysis of mitochondrial apoptotic pathway related molecules. When MT-2 cells were cultured with 25 µg/ml of CA, [A] cytochrome-c release to Cy (cytoplasma) from Mi (mitochondria), [B] increase of the Bax/Bcl2 balance, and [C] phosphorylation of apoptosis-stimulating MAP kinases such as JNK and p38 were observed.

5 A B Cell number O 2 - (hydroethidine) Positive for O 2 - (hydroethidine) (%) Fig. 4 Production of superoxide in MT-2 cells cultured with CA. [A] Production of superoxide was detected by flow cytometry and [B] The cell percentage of positive for O - 2 cells increased in a dose dependent manner.

6 Since cellular environmental stress or cytotoxic chemical substances induce activation of the mitochondrial apoptotic pathway to cause cell death, the role of ROS (reactive oxygen species) is considered a very important factor. Therefore, the production of superoxide was analyzed using flow cytometry As shown in Fig. 4, MT-2 cells cultured with, 1, 25, and 5 µg/ml of CA showed increased production of superoxide in a dose dependent manner. Together with data showing that anti-oxides significantly reduced CA-induced apoptosis in MT-2 cells, these results indicate that MT-2 cells undergo apoptosis via activation of the mitochondrial pathway when cells are exposed to chrysotile for a short time and in high doses. Establishment of an In Vitro T Cell Model For Long-Term and Low Dose Exposure to Asbestos As mentioned above, MT-2 cells proceeded to apoptosis when cultured with 25 to 5 µg/ml of chrysotile for a few days. We considered that if MT-2 cells were continuously exposed to relatively low doses (i.e., 5 or 1 µg/ml) of chrysotile for months to years, these cells might acquire resistance to chrysotileinduced apoptosis. Although the initial cultures of MT-2 cells with 5 or 1 µg/ml of chrysotile-b (CB) only permitted us to change the whole medium and to keep total cell number in their culture dishes, several months later, they gradually started to grow even with low doses of CB. Eight months after starting with low-dose continuous exposure, the exposed subline (MT-2Rst) showed a decrease in the appearance of TUNEL positive fractions when cultured with high doses (25 to 5 µg/ml) of CB when compared with the original MT-2 cells (MT-2Org). In addition, the growth of MT- 2Rst cultured with high doses of CB was not reduced when assayed by WST-1 (Fig. 5A and B). These results indicated that more than eight months exposure to CB induced resistance to chrysotile-induced apoptosis in MT-2 cells. The cellular and molecular biological differences between MT-2Org and MT-2Rst should be analyzed to try to determine the mechanisms involved in the silicate/silica induced dysregulation/reduction of the human immune system. As a first step, cdna microarray analysis was employed to find gene expression differences between these two lines. In Tables 1 and 2, the up- and down-regulated genes are respectively listed in the order of their relative expression ratio (MT-2Org/MT-2Rst) in the gene. Among these genes, there are several genes of interest, such as the chloride channel 5 gene (clcn5), one of the chemokines (scya8), and one of the virus receptors (cxadr) in the upregulated group, and one of the chemokines (scyb13) and the receptor type protein tyrosine phosphatase (ptprn2; this is also an autoantibody found in type I DM patients) in the downregulated group.

7 A Proliferation ratio (% of control) detected by WST-1 assay Concentration of Chrysotile-B (µg/ml) Original Resistant MT-2 cells subline cells B Resistant subline cells (1) Original MT-2 cells TdT(-) control µg/ml Fig. 5 MT-2, showed dose-dependent growth inhibition [A, left] and a dose-dependent increase in TUNEL-positive apoptotic cells [B, upper panel], when cultured with various concentrations of Chrysotile-B (CB) for two days. Ho wever, after continuous exposure to 1 µg/ml of CB for eight months, a CB-resistant subline, which showed no CB-induced growth inhibition [A, right] and the appearance of an apoptotic fraction [B, lower panel], were observed.

8 Table 1 The list of up-regulated genes in the MT-2-Rst line compared with the MT-2-Org line analyzed by cdna microarray Name of genes Ratio: Original/Resistant subline poly(a) polymerase gamma; papolg.91 chloride channel 5; clcn5.124 tolloid-like 2; tll2.162 coxsackie virus and adenovirus receptor; cxadr.179 epoxide hydrolase 1, microsomal (xenobiotic); ephx1.22 small inducible cytokine subfamily a (cys-cys), member 8 (monocyte chemotactic protein 2); scya8.22 chromobox homolog 8; cbx8.28 similar to non-functional folate binding protein loc ebv-induced g protein-coupled receptor 2; ebi2.31 doublecortex; lissencephaly, x-linked (doublecortin); dcx.35 chromosome 2 open reading frame 1; c2orf1.358 nhp2 non-histone chromosome protein 2-like 1 (s. cerevisiae); nhp2l1.378 potassium voltage-gated channel, shaw-related subfamily, member 1; kcnc1.391 mad, mothers against decapentaplegic homolog 3 (drosophila); madh3.412 chromosome 8 open reading frame 1; c8orf1.414 pro2353 predicted protein of hq

9 Name of gene Table 2 The list of downregulated genes in the MT-2-Rst line compared with the MT-2-Org line analyzed by cdna microarray Ratio:Original/Resistant subline small inducible cytokine b subfamily (cys-x-cys motif), member 13 (b-cell chemoattractant); scyb protein tyrosine phosphatase, receptor type, n polypeptide 2; ptprn eukaryotic translation initiation factor 4a, isoform 1; eif4a cgi-143 protein; loc nadh dehydrogenase (ubiquinone) 1 beta subcomplex, 6 (17kd, b17); ndufb ubiquitin-conjugating enzyme e2n (homologous to yeast ubc13); ube2n hypothetical protein xp_5115; loc karyopherin (importin) beta 3; kpnb chaperonin containing tcp1, subunit 4 (delta); cct proteasome (prosome, macropain) subunit, alpha type, 1; psma non-metastatic cells 1 protein; nme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mrna, complete cds; glyceraldehyde-3 phosphate dehydrogenase (EC ) tata box-binding protein-associated factor 2f; taf pituitary tumor-transforming protein 1; pttg h2a histone family, member z; h2afz 2.81 testican 3; hsaj cofilin 1 (non-muscle); cfl ran binding protein 1; ranbp splicing factor, arginine/serine-rich (transformer 2 drosophila homolog) 1; sfrs signal recognition particle 14kd (homologous alu rna binding protein); srp tryptophanyl-trna synthetase; wars fumarate hydratase; fh 2.65 methionyl aminopeptidase 2; metap hypothetical protein flj1156; flj trp-related cation influx channel; trpm transcription elongation factor a (sii), 1; tcea

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